1. Field of the Invention
The present invention relates to the production of human parathyroid hormone (hereinafter referred to as “hPTH”). More particularly, the present invention relates to the use of Saccharomyces cerevisiae mutant strains which are genetically disrupted in at least one of the yapsin family of aspartic proteases YPS1, YPS2 and YPS3 and transformed with an expression vector anchoring a hPTH gene, in producing the hormone.
2. Description of the Prior Art
hPTH is a peptide consisting of 84 amino acid residues, produced by the parathyroid gland. hPTH maintains calcium homeostasis in the kidneys and bones, having the physiological function of promoting calcium metabolism and osteogenesis. In the U.S.A. and the Europe, estrogen or calcitonin have been predominantly used as therapeutics for osteoporosis, but are found to be obstructive of bone absorption. Accordingly, leading pharmaceutical companies of the world are intensively and extensively studying hPTH for the development of therapeutics for osteoporosis by taking advantage of the ability of hPTH to promote osteogenesis. Particularly, more earnest attention is paid to the prophylaxis and treatment of osteoporosis as modern society becomes an aging society. For above reasons, active research is now being directed to the development of biotechnology methods for the mass production of hPTH, which is expected to be a promising therapeutic substitutive for conventional therapeutics for osteoporosis.
For example, various attempts have been made to develop methods for mass-producing recombinant hPTH by using E. coli as a host cell because the prokaryotic bacteria shows relatively high expression efficiency. However, there is a great difficulty in refolding and purifying the recombinant proteins produced from E. coli. In contrast, yeast, a single cell eukaryote, has the advantage of expressing and secreting properly folded- and thus active-proteins because it is very similar to higher species in the gene transcription and translation systems, and its protein-secretion system. Additionally, yeast is advantageous as a host for producing proteins of interest in that yeast secretes few extracellular proteins making it easy to recover and purify exogenous proteins. Further, the yeast Saccharomyces cerevisiae is a GRAS (generally recognized as safe) microorganism that is not pathogenic to the body and does not produce endotoxins. With the anticipation of being very useful as a producer of medicinal recombinant hPTH, Saccharomyces cerevisiae has been studied in developing hPTH expression systems. In spite of its various advantage as an expression host for the production of recombinant hPTH, Saccharomyces cerevisiae is not industrially utilized as such a host because the extracellularly secreted recombinant hPTH is degraded by endogenous proteolytic enzymes of the yeast's own, so that only a small amount of the intact molecule of hPTH can be recovered (Gabrielsen et al., Gene 90, 255(1990)).
In the last decade, extensive studies have been made for solving the proteolysis problem of recombinant hPTH. For instance, based on the finding that hPTH is cleaved between Arg-25 and Lys-26, which is identical to the recognition site of KEX2, a protease present in yeast Golgi bodies, a substitution mutant of hPTH, which has glutamine at position 26 of its amino acid sequence instead of lysine, was made with the aim of preventing the proteolysis by KEX2, which is a putative protease to cleave hPTH (Reppe et al., J. Biol. Chem. 266, 14198 (1991)). Using gene recombination technology, an hPTH-related protein was directly ligated to the 3′-end of the yeast ubiquitin gene to produce a non-cleavable hPTH-related protein (Rian et al., Eur. J. Biochem., 213, 641 (1993)). However, if protein mutants are medicinally used, there are required stringent tests to obtain permission for their medicinal use, because they are generally recognized as new medicines. When using gene fusion technology, it is necessary to remove the fusion site by expected digestion because proteins of interest may be produced at relatively yields owing to the presence of the fusion site.
As a result of the research for the prevention of hPTH degradation without resorting to hPTH protein mutants or fusions, the present inventors developed a method in which hPTH cleavage can be prevented to a significant extent simply by adding L-arginine at high concentrations to the culture media (Chung and Park, Biotechnol. Bioeng. 57, 245 (1998)). The fact that hPTH cleavage is prevented to a significant extent by the presence of L-arginine in the culture media indicates that hPTH cleavage is performed mainly by extracellular proteases rather than by the intracellular protease Kex2p. From this finding, the present inventors inferred that Yap3p (yeast aspartic protease 3), which, like KEX2, is able to cleave the C-terminal or middle sites of basic single or couple amino acids, and binds to the cytoplasmic membrane of yeast, is practically responsible for the cleavage of the hPTH secreted into yeast culture media. On the basis of this inference, the present inventors made a YAP3 gene-disrupted yeast strain (yap3Δ), and constructed an hPTH production system by use of the yeast mutant. Upon culturing in flasks, the hPTH production system was found to prevent the cleavage of hPTH at an efficiency of as high as 80%, thus producing the intact molecule of hPTH at high yield (Kang et al., Appl. Microbiol. Biotechnol., 50, 187 (1998); Korean Pat. No. 0246932, yielded Dec. 8, 1999). Though exhibiting far higher hPTH productivity than the wild type strain, the yap3Δ mutant was observed to allow hPTH to be cleaved to a significant extent in the late stage of the high-concentration culturing (Song and Chung, Process Biochem 35, 503 (2000)), which indicates that hPTH is cleaved not by Yap3p, but by other proteases in the late culture stage. Saccharomyces cerevisiae is reported to have the aspartic protease MKc7p, which is very similar in structure and function to Yap3p (Komano and Fuller, Proc Natl Acad Sci, USA, 92, 10752 (1995)). The present inventors created a yeast mutant in which both of the genes are disrupted (yap3Δ/mkc7Δ) for use in the observation of the influence of the enzymes on the hPTH cleavage at the terminal culture stage. However, no significant differences were found between the mutants yap3Δ and yap3Δ/mkc7Δ (Choi, et al., J. Microbiol Biotechnol 9, 679 (1999)). These results demonstrated that the MKc7p protease, through having high homology with Yap3p (53% homology) and being involved in the processing of pro-α-mating factor in the absence of Kex2p, is not greatly responsible for hPTH cleavage.
Through the analysis of the recently disclosed genome information about Saccharomyces cerevisiae, a search was made for genes homologous to YAP3 (recently renamed YPS1), resulting in the finding that three novel genes coding for unknown aspartic proteases, in addition to PEP4 and BAR1, are present (Olsen et al., Biochem. J. 339, 407 (1999)). Assumed to encode new members of the yapsin family of aspartic proteases, like YPS1 and YPS2, the three novel genes were named YPS3, YPS6 and YPS7, respectively. YPS3 was found to have 50% homology with both of YPS1 and YPS2, while 35% and 25% homology was found between YPS6 and BAR1 and between YPS7 and PEP4, respectively. From the homology between YPS3 and YPS1, the present inventors drew the deduction that the hPTH cleavage occurring at the terminal culture stage of the yps1Δ (previous yap3Δ) strain might be performed by yapsin 3.